| Part 1 The role of fosinopril and valsartan intervention in Klotho gene,MMP-9,TIMP-1,and PAI-1 gene expression in SHR kidneysObjective The role of fosinopril and valsartan intervention in spontaneously hypertensive rats(SHR)on Klotho MMP-9,TIMP-1 and PAI-1 expression in hypertensive renal interstitial fibrosis in kidney tissue of SHR.Methods 20 male 22-week-old SHR(spontaneously hypertensive rats)22-week-old,were randomly divided into four groups(n=5): following 8 weeks of continuous administration,hypertension group (SHR group),fosinopril group(Fos group,10 mg/Kg/d gavage),valsartan group(Val group,10 mg/Kg/d gavage)and fosinopril plus valsartan group(Fos+Val group,fosinopril 10 mg/Kg/d+valsartan 50 mg/Kg/d gavage);22-week-old male Wistar Kyoto rats(WKY)as controls. Through monitoring of weight of the rats,tail artery pressure,24-hour urine protein by fosinopril and/or valsartan intervention after 8 week trial, HE staining of renal pathological changes,immunohistochemical and Western Blot detection of Klotho,MMP-9,TIMP-1 and PAI-1 protein expression in the kidneys,RT-PCR detection of renal tissue Klotho, MMP-9,TIMP-1 and PAI-1 mRNA expression,analysis of the results was performed. 1.At the end of the 30 weeks the experiment showed that the systolic blood pressure and serum creatinine in SHR group,were significantly higher than those in WKY group(P＜0.05).As for the fosinopril and / or valsartan after treatment,Fos group,Val group and Fos +Val group rats,systolic blood pressure and serum creatinine were significantly lower than those in SHR group(P＜0.05),Meanwhile,The experiments before and after the eight week trial yielded,urinary protein excretion difference(△UPro)higher in Fos group,Val group and Fos +Val group than those in SHR group(P＜0.05).2.HE staining of renal pathological changes seen in hypertensive renal damage in SHR group,and fosinopril and/or valsartan intervention significantly improved renal damage.3.By virtue of Immunohistochemical results,the expression of Klotho,MMP-9,TIMP-1 and PAI-1 protein in the cytoplasm of both tubules was clearly demonstrated in WKY group.In SHR group,Klotho protein expression was significantly decreased,whilst MMP-9, TIMP-1,PAI-1 protein expression were significantly higher compared to the WKY group.In the case of fosinopril and/or valsartan intervention, Klotho protein expression was increased in Fos group,Val group(P＜0.01)and Fos+Val group(P＜0.05),while MMP-9(all P＜0.01), TIMP-1 protein expression(all P＜0.05)were decreased in Fos group,Val group and Fos+Val group,while PAI-1 protein expression was decrease in Fos group(P＜0.01),Val group and Fos+Val group(all P＜0.05).4.The results of RT-PCR showed that in the SHR group,Klotho mRNA expression was significantly decreased,whilst MMP-9,TIMP-1, PAI-1 mRNA expression were significantly higher compared to the WKY group,with fosinopril use and/or valsartan intervention,Klotho mRNA expression in Fos group and Fos+Val group(all P＜0.01),Val group(P＜0.05)was significantly increased compared with those in SHR group, whereas MMP-9,TIMP-1 and PAI-1 mRNA expression in Fos group,Val group and Fos+Val group was significantly lower than those in SHR group(all P＜0.01).Correlation analysis demonstrated that the Klotho mRNA expression of MMP-9,TIMP-1,PAI-1(r=-0.864,respectively, -0.725 and -0.785,P＜0.01)expression showed a negative correlation.5.The results of western blot showed that in the case of fosinopril use and/or valsartan intervention after 8 weeks,Klotho protein expression in Fos group(P＜0.05),Val group and Fos+Val group(all P＜0.01)increased compared to the SHR group,While MMP-9,TIMP-1 and PAI protein expression in the Fos group,Val group and Fos+Val group was significantly lower than those in SHR group(P＜0.01). Correlation analysis showed that the Klotho protein expression of protein (r=-0.614,respectively,-0.579 and -0.522,P＜0.05)expression showed a negative correlation.Conclusions:1.In the SHR model of renal tissue,Klotho mRNA and protein expression declined while MMP-9,TIMP-1 and PAI-1 mRNA and protein expression increased,which can be reversed by fosinopril and / or valsartan.2.Klotho gene expression and MMP-9,TIMP-1 and PAI-1 gene expression in renal tissue were negatively correlated,suggesting that Klotho gene expression and the gene related to extracellular matrix degradation are closely related.3.The role of fosinopril and valsartan in resistance to renal fibrosis may involve altering gene expression of Klotho gene and MMP-9,TIMP-1 and PAI-1 gene,and the biological effect of anti-aging gene Klotho may be achieved through downstream target gene express of MMP-9,TIMP-1 and PAI-1 gene. Part 2 Klotho,MMP-9,TIMP-1 and PAI-1 gene expression and the effect of fosinopril and valsartan intervention in human renal tubular epithelial cellsObjective To study the angiotensinⅡ(AngⅡ)stimulation of human HK-2 renal tubular epithelial cells and Klotho,MMP-9,TIMP-1 and PAI-1 gene expression to explore the impact of fosinopril and vasartan intervention.Method The HK-2 human renal tubular epithelial cells and cultures set for assessment of drag intervention were divided into four groups:angiotensinⅡgroup(Ang group,10-7mol/L),Fosinopril group (Fos group,fosinopril 10-5mol/L+angiotensinⅡ10-7mol/L),Valsartan group(Val group,valsartan 10-5mol/L+ angiotensinⅡ10-7mol/L),Fos+ Val group(fosinopril 10-5mol/L+valsartan 10-5mol/L+angiotensinⅡ10-7mol/L);another HK-2 cell line as a normal control group(Control group).Using immunohistochemistry and Western Blot techniques, Klotho,MMP-9,TIMP-1 and PAI-1 protein expression were determined in HK-2.Using RT-PCR in HK-2,the Klotho,MMP-9,TIMP-1 and PAI-1 mRNA expression were determined and correlation analysis performed.Results:1.Immunohistochemistry of the results showed that in HK-2,AngⅡcan inhibit the expression of the Klotho mRNA and protein whilst promoting MMP-9,PAI-1 and TIMP-1 mRNA and protein expression,in the case of fosinopril and / or valsartan intervention,Klotho protein expression was increased while MMP-9 and TIMP-1 protein expression was reduced(all P＜0.05)in Fos group,Val group and Fos+Val group (all P＜0.05),PAI-1 protein expression in Fos group(P＜0.01),Val group and Fos+Val group(all P＜0.05)was decreased.2.The results of RT-PCR showed that with fosinopril and /or valsartan intervention,Klotho mRNA expression was increased while PAI-1 mRNA expression was decreased in Fos group(all P＜0.05),Val group and Fos+Val group(all P＜0.01).Correlation analysis showed a negative correlation between Klotho mRNA expression with MMP-9, TIMP-1,PAI-1 mRNA(r-0.698 and-0.835,respectively,-0.644,P＜0.01).3.Western blot results showed that fosinopril and/or valsartan promotes AngⅡ-induced downregulation of Klotho protein expression (P＜0.01)and inhibits AngⅡ-induced upregulation of MMP-9,TIMP-1 (all P＜0.01)in Fos group,Val group and Fos+Val group(all P＜0.01)and inhibits AngⅡ-induced upregulation of PAI-1 protein in Fos group (P＜0.05),Val group and Fos+Val group(all P＜0.01).Correlation analysis showed a negative correlation between Klotho protein expression with MMP-9,TIMP-1,PAI-1 protein expression(r-0.690,respectively, -0.823 and-0.646,P＜0.01).Conclusion:1.Normal HK-2 cells show definite Klotho,MMP-9,TIMP-1 and PAI-1 mRNA and protein expression.2.In HK-2 cells,AngⅡinduces MMP-9,TIMP-1 and PAI-1 mRNA and protein expression,suggesting that AngⅡcan affect MMP-9,TIMP-1 and PAI-1 mRNA and protein expression,thereby affecting ECM degradation.AngⅡinhibition of Klotho mRNA and protein expression, Klotho and MMP-9,TIMP-1 and PAI-1 gene expression were negatively correlated,positing AngⅡas a major effector peptide of RAS system which plays an important role in express of anti-aging gene Klotho and the gene related with extracellular matrix degradation in renal tubular epithelial cells.The MMP-9,TIMP-1 and PAI-1 gene expressions may be closely related to renal interstitial fibroses.3.Fosinopril and /or valsartan block the AngⅡin HK-2 cells, thereby increasing Klotho mRNA and protein expression and decreasing MMP-9,TIMP-1 and PAI-1 mRNA and protein expression,suggesting that the contribution of ACEI and/or ARB in decreasing the level of AngⅡmay be an important mechanism of interstitial fibrosis through control of gene expression of Klotho,MMP-9,TIMP-1 and PAI-1. Conclusion1.The in vivo and in vitro study results showed that in the SHR model of renal tissue,Klotho gene expression decreased while MMP-9, TIMP-1 and PAI-1 gene expression increased,and correlation analysis showed a negative correlation between the express of Klotho gene and MMP-9,TIMP-1,PAI-1 gene was negatively correlated,positing anti-aging gene and the genes related with extracellular matrix degradation as important players in hypertensive renal injury.2.The expression of Klotho,MMP-9,TIMP-1,PAI-1 in SHR model of renal tissue and hypertensive renal injury can be reversed by fosinopril and valsartan.3.The expression of Klotho gene and MMP-9,TIMP-1,PAI-1 gene was negatively correlated,which showed that Klotho-induced alteration of MMP-9,TIMP-1 and PAI-1 gene expression can control extracellular matrix degradation and which can improve renal interstitial fibrosis brought about by hypertensive renal injury.4.The contribution of fosinopril and valsartan to interstitial fibrosis is conjectured to arise through altering gene at the Klotho-MMPs/TIMPs-PAI-1 level. |
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